Polyhydroxyalkanoates and methods of making thereof

Abstract

Provided are microorganisms for making polyhydroxylalkanoate (PHA) compounds. For instance, the microorganism can include a polyhydroxylalkanoate (PHA) synthase (phaC) gene and one or both of an isocaprenoyl-CoA:2-hydroxyisocaproate CoA-transferase (hadA) gene and a propionate CoA-transferase (pct) gene. In some cases, the species of the microorganism is a Cupriavidus necator bacteria that has been genetically modified to include the PHA and hadA or pct genes.

1 . A genetically modified microorganism comprising:
 a heterologous polyhydroxyalkanoate (PHA) synthase (phaC) gene; and   an isocaprenoyl-CoA:2-hydroxyisocaproate CoA-transferase (hadA) gene and/or a propionate CoA-transferase (pct) gene,   wherein the microorganism is capable of producing a PHA polymer.   
     
     
         2 . The microorganism of  claim 1 , wherein the species of the microorganism is  Cupriavidus necator.    
     
     
         3 . The microorganism of  claim 2 , wherein the  Cupriavidus necator  is a ΔphaC1 mutant of  Cupriavidus necator.    
     
     
         4 . The microorganism of  claim 1 , wherein the phaC gene has at least 80% sequence identity to a PHA synthase (phaC) gene from a bacterium of the  Pseudomonadaceae  genus. 
     
     
         5 . The microorganism of  claim 4 , wherein the bacterium of the  Pseudomonadaceae  genus is  Pseudomonas  sp. MBEL 6-19. 
     
     
         6 . The microorganism of  claim 1 , wherein the isocaprenoyl-CoA:2-hydroxyisocaproate CoA-transferase (hadA) gene has at least 80% sequence identity to a gene from  Clostridium difficile,  wherein the microorganism comprises the hadA gene. 
     
     
         7 . The microorganism of  claim 1 , wherein the propionate CoA-transferase (pct) gene has at least 80% sequence identity to a gene from  Clostridium propionicum,  wherein the microorganism comprises the pct gene. 
     
     
         8 . The microorganism of  claim 1 , wherein the PHA polymer comprises a carbon atom metabolized from a carbon source by the microorganism. 
     
     
         9 . The microorganism of  claim 8 , wherein the carbon source is selected from the group consisting of: a gaseous mixture comprising CO 2  and H 2 , formic acid, acetic acid, fructose, sucrose, or salts thereof. 
     
     
         10 . The microorganism of  claim 1 , wherein the PHA polymer has the formula (I): 
       
         
           
           
               
               
           
         
         wherein: 
         n and m define the mol % of each unit within the PHA polymer, wherein n ranges from greater than 0% to 100% and m is 100% minus n. 
         X 1  and X 3  are each independently absent, arylene, heteroarylene, substituted arylene, or substituted heteroarylene; and 
         X 2  and X 4  are each independently alkylene, alkenylene, alkynylene, arylene, heteroarylene, substituted alkylene, substituted alkenylene, substituted alkynylene, substituted arylene, or substituted heteroarylene. 
       
     
     
         11 . A method of making a polyhydroxyalkanoate (PHA) polymer of formula (I): 
       
         
           
           
               
               
           
         
         wherein: 
         n and m define the mol % of each unit within the PHA polymer, wherein n ranges from greater than 0% to 100% and m is 100% minus n. 
         X 1  and X 3  are each independently absent, alkylene, arylene, heteroarylene, substituted arylene, or substituted heteroarylene; and 
         X 2  and X 4  are each independently alkylene, alkenylene, alkynylene, arylene, heteroarylene, substituted alkylene, substituted alkenylene, substituted alkynylene, substituted arylene, or substituted heteroarylene, 
         the method comprising the step of culturing a microorganism to produce the PHA polymer of formula (I). 
       
     
     
         12 . The method of  claim 11 , wherein the microorganism is a microorganism according to  claim 1 . 
     
     
         13 . The method of  claim 11 , wherein the culturing comprises contacting the microorganism with a carbon source and the PHA polymer comprises a carbon atom from the carbon source. 
     
     
         14 . The method of  claim 13 , wherein the carbon source is selected from the group consisting of: a gaseous mixture comprising CO 2  and H 2 , formic acid, acetic acid, fructose, sucrose, and salts thereof. 
     
     
         15 . The method of  claim 14 , wherein the carbon source is a gaseous mixture comprising CO 2  and H 2 . 
     
     
         16 . The method of  claim 11 , wherein m is greater than 0% and wherein the culturing comprises contacting the microorganism with a compound of formula (II): 
       
         
           
           
               
               
           
         
         or a salt thereof. 
       
     
     
         17 . The method of  claim 16 , wherein the compound of formula (II) is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         and salts thereof. 
       
     
     
         18 . The method of  claim 11 , wherein X 1  is absent and X 2  is alkylene or substituted alkylene. 
     
     
         19 . The method of  claim 18 , wherein the “n” monomer has the structure of N1: 
       
         
           
           
               
               
           
         
         or a stereoisomer thereof. 
       
     
     
         20 . The method of  claim 11 , wherein m is greater than 0% and X 3  is absent. 
     
     
         21 . The method of  claim 20 , wherein X 4  is alkylene or substituted alkylene. 
     
     
         22 . The method of  claim 21 , wherein the “m” monomer has the structure of M1 or M2: 
       
         
           
           
               
               
           
         
         or a stereoisomer thereof. 
       
     
     
         23 . The method of  claim 11 , wherein m is greater than 0% and X 3  is arylene or substituted arylene. 
     
     
         24 . The method of  claim 23 , wherein X 4  is alkylene or substituted alkylene. 
     
     
         25 . The method of  claim 24 , wherein the “m” monomer has the structure of M3: 
       
         
           
           
               
               
           
         
       
     
     
         26 . The method of  claim 11 , wherein X 3  is alkylene and X 4  is heteroarylene. 
     
     
         27 . The method of  claim 26 , wherein the “m” monomer has the structure of M4: 
       
         
           
           
               
               
           
         
       
     
     
         28 . A compound of formula (I): 
       
         
           
           
               
               
           
         
         wherein: 
         n and m define the mol % of each unit within the PHA polymer, wherein n ranges from greater than 0% to 100% and m is 100% minus n. 
         X 1  and X 3  are each independently absent, alkylene, arylene, heteroarylene, substituted arylene, or substituted heteroarylene; and 
         X 2  and X 4  are each independently alkylene, alkenylene, alkynylene, arylene, heteroarylene, substituted alkylene, substituted alkenylene, substituted alkynylene, substituted arylene, or substituted heteroarylene. 
       
     
     
         29 . The compound of  claim 28 , wherein the “n” monomer has the structure of N1 and the “m” monomer has the structure M3: 
       
         
           
           
               
               
           
         
       
     
     
         30 . The compound of  claim 28 , wherein the “n” monomer has the structure of N1 and the “m” monomer has the structure M4: